"Transgene" or "exogenous gene" are terms used in the art to denote a gene which has been transferred to a host cell or host plant from a source other than the host cell or host plant. However, a transgene may herein refer to a gene normally locatable in an host plant or plant cell, to which is added one or more flanking regions comprising stabilizing DNA segments. Thus a gene endogenous to a host plant cell or host plant may be modified with flanking regions comprising stabilizing DNA segments whereby the endogenous gene becomes an exogenous gene or transgene with the meaning of the instant application. As used herein, the terms "transgene" and "exogenous gene" have the same meaning. Transfer of an exogenous gene to a host plant cell can be accomplished by a variety of means known in the art. Most classes of plants have been transformed and regenerated to yield adult plants expressing a phenotype associated with the transgene.
The process of producing a transgenic plant typically includes exposing plant cells, which may be in the form of individual cells, protoplasts or excised tissue, to DNA comprising the exogenous gene, in order to introduce the exogenous gene into the host cells. Only a fraction of the cells exposed to the DNA are actually transformed. The recipient cells are then cultured in vitro in order to proliferate the transformants and to identify and select for those which express the transgene. Frequently a selectable marker is introduced, together with the exogenous DNA, so that transformants can be selected by their ability to grow under conditions that inhibit growth of nontransformed cells, or which favor growth of transformed cells. However, it is also possible in some cases to identify directly the transformed cells or callus containing them. Further steps include techniques of regeneration to produce differentiated shoots, roots or embryos from which, ultimately, whole plants can be obtained. For reviews on plant transformation and regeneration, see Crossway, A. et al. (1986) Mol. Gen. Genet. 202:179-185; Horsch, R. B. et al. (1985) Science 227:1229-1231; Gasser, C. S. and Fraley, R. T. (1989) Science 244:1293-1299; Marton, L. et al. (1979) Nature 277:129-131; Klein, T. M. et al. (1988) Proc. Natl. Acad. Sci. USA 85:8502-8505; Krens, F. A. (1982) Nature 296:72-74; Deshayes, A. et al. (1985) EMBO J. 4:2731-2739; Deroles, S. C. and Gardner, R. C. (1988) Plant Mol. Biol. 11:365-377; Czernilofsky, A. P. et al. (1986) DNA 5:101-113; Hain, R. et al. (1985) Mol. Gen. Genet. 199:161-168; Scheerman, S. and Bevan, M. W. (1988) Plant Cell Reports 7:13-16; Shillito, R. D. et al. (1965) Bio/Technology 3:1099-1102; Valvekens, D. et al. (1988) Proc. Natl. Acad. Sci. USA 85:5536-5540.
Primary transformants are those cells or proliferated tissue (e.g., callus colonies) which are initially observable, directly or indirectly, as possessing the exogenous gene after the transformation step. Most commonly, possession of the exogenous gene is observed indirectly as expression of a co-transformed selectable marker. In some instances the phenotype associated with expression of the exogenous gene will be observable in the primary transformant. When a selectable marker is present, such as antibiotic resistance, culture in the presence of the selection agent, the antibiotic, ensures that only cells expressing the resistance phenotype will grow. In the absence of selection, however, it has frequently been observed that descendants of primary transformants lose the phenotype associated with the transgene. For example, explants of a primary transformant callus often fail to display the phenotype of the exogenous gene, in the absence of continued selection pressure. Furthermore, when whole plants are regenerated from transformed tissue or callus, some of the regenerated plants fail to have the phenotype of the exogenous gene, and the same phenomenon is sometimes observed in the progeny of selfed transformed plants. In such cases it has not been established whether the loss of phenotype is due to loss of the exogenous gene itself, or to loss of ability to express the exogenous gene. The loss of phenotype, whatever the mechanism, results in a gradual decline in overall transformation efficiency, i.e., the total number of transformants declines over time with respect to the number of initial transformants. The present invention provides a means for stabilizing transformants against loss of phenotype associated with the exogenous gene, so that higher overall transformation efficiency is obtainable.
Recent studies of the structure of the eucaryotic cell nucleus and the organization of chromatin within the nucleus have led to new techniques for identifying DNA components and nuclear structural components that participate in organizing cellular DNA in the nucleus. Such studies have demonstrated the existence of a complex nuclear matrix which includes structural components remaining after DNAse I digestion and extraction with 2M NaCl (See Gasser, S. M. (1988) Architecture of Eukaryotic Genes: Symposium on Chromatin Structure of Plant Genes, Frankfurt am Main, W. Germany, September 1986, XIV+518, P VCH Publishers; New York, pp. 461-471). The finding that Li-3,5-diiodosalicylate (LIS) extraction removes histone from chromosomal DNA has made it possible to isolate nuclear scaffold by combining LIS extraction with endonuclease digestion. Such procedures leave residual DNA segments bound to the nuclear scaffold. Such DNA segments have been termed scaffold attachment regions (SAR) and matrix associated regions (MAR). Such DNA segments are considered to be functionally similar in nature, independent of how they are obtained. The term MAR is used herein to refer to DNA segments isolated from nuclear scaffold or nuclear matrix preparations after endonuclease treatment.
MARs typically bind reversibly to nuclear matrix or scaffold preparations. Binding is saturable, indicating binding to a limited number of specific sites. MARs can be of any size, however, they are generally of about 1 kb or less in size and are generally AT rich. They do not necessarily share extensive sequence homology, although certain sequence motifs have been observed in some MARs. Many MARs possess a topoisomerase II cleavage site consensus sequence.
MARs are believed to function in vivo as structural attachment points linking chromosomal DNA to structural elements of the nucleus. Models of chromosome structure have been proposed, in which chromosomal regions between two adjacent MARs form a loop of DNA between the anchor points of the MARs. It has been proposed that MAR attachment facilitates transcription of nearby genes, by locating those genes close to nuclear pores or channels where polymerases, transcription factors, substrates, etc., may concentrate. At the same time, anchorage to the nuclear matrix serves to separate or isolate groups of genes on separate chromatin loops, acting as boundary elements to limit the influence of nearby transcription units on one another, commonly called position effects.
The MARs appear to function across species boundaries, although matrix binding specificity may be diminished, for example when using animal MARs in plants. Functional association between MARs and DNA replication has also been implicated in studies showing that some matrix-binding sequences of maize DNA may function as ARS (autonomous replicating sequence) elements in yeast.
Phi-Van, L. et al. (1990) Mol. Cell Biol. 10:2302-2307, compared the effect on reporter gene expression of the presence or absence of MARs flanking an exogenous reporter gene. The MAR sequence was isolated from the chicken lysozyme gene 5' flanking region, the host cells were fibroblasts. Both enhancement of reporter gene activity and reduction of position effects (individual variation of expression level among independent transfectants) were observed if the reporter gene construct included MARs flanking the gene. A review of boundary functions attributable to MARs was published by Eissenberg, J. C. and Elgin, S. C. R. (1991) Trends in Genetics 7:335-340. The authors suggested that MARs function both as insulators when bracketing a gene together with its enhancers, to maintain activity of the enhancers by isolating them from chromosomal position effects, and as barriers when interposed between a gene and an enhancer.
In higher plants, the existence of MARs has been reported by Hall, G et al. (1991) Proc. Natl. Acad. Sci. USA 88:9320-9324. Tobacco MARs (termed SARS therein) were isolated from flanking regions of three root-specific genes. An "endogenous" assay for MARs was disclosed, based on their ability to bind to nuclear scaffold preparations. An "exogenous" assay, based on ability of isolated scaffolds to bind DNA fragments containing MARs, was also disclosed. A scaffold-associated DNA region located downstream of the pea plastocyanin gene was isolated and characterized by Slatter, R. E. et al. (1991) Plant Cell 3:1239-1250. The SAR was linked to a downstream repeated sequence and had a sequence rich in A and T sequences, several topoisomerase II binding sites and several ARS sequences.
Breyne, P. et al. (1992) Plant Cell 4:463-471 used a tobacco-derived SAR to analyze the effect of flanking a reporter gene in transgenic plants, similar to the experiment described by Phi-Van et al. (1990). Qualitatively similar results were obtained showing an effect of a tobacco SAR flanking a transgene on variance of expression among independent tobacco transformants. However, no increase of average expression level was observed. The effect of reducing variance was not observed for constructs containing a mammalian .beta.-globin SAR instead of the tobacco SAR. All effects heretofore observed have related to phenomena occurring within a single generation.